The agar culture system newly developed in this Unit for cloning mouse multipotential hemopoietic stem cells offers the first feasible method for duplicating and analysing in vitro the mechanisms by which acute and chronic myeloid leukemia develop in man. The system also permits an analysis of the induction steps by which these multipotential cells generate committed hemopoietic progenitor cells. These colonies are derived from single initiating cells (MIX-CFC), average 15,000 cells in size and are composed of erythroid, neutrophil, macrophage, eosinophil and megakaryocytic cells. Proliferation of colony cell requires stimulation by pokeweed mitogen-stimulated spleen conditioned medium (SCM) that has been shown to contain glycoprotein regulatory factors. It is proposed to transform CBA fetal liver MIX-CFC in vitro using irradiation, Friend or Abelson virus and/or methylnitrosourea in attempts to develop a clonal model of leukemia induction in vitro in which only one subpopulation of the clonal progeny is transformed. Transformation will be monitored by recloning in vitro and by transplantation of pooled or individual transformed colonies to normal or preirradiated CBA T6T6 mice. The influence of excess levels of purified granulocyte-macrophage colony stimulating factor (GM-CSF), purified erythropoietin and SCM on the transformation will be determined. The specific regulatory factors in SCM will be further purified and their role in stem cell commitment analysed in normal and transformed colonies by recloning of individual developing colony cells. Attempts will be made using purified hemopoietic regulators to induce hemopoietic differentiation in vitro in 4 types of undifferentiated tumor to further analyse the role of regulatory factors in leukemia induction.